GABRB2基因羟甲基化修饰与精神分裂症的相关性研究

Impairments of GABAergic transmission induced by genetic, epigenetic or stress factors underlie the pathophysiology of schizophrenia (SZ). Molecular mechanisms such as epigenetic modifications mediating the interplay between genetic and stress factors display a significant role in the onset of this disease, but the detailed mechanism remains to be elucidated. Our recent works found that RNA expression of the type A gamma-aminobutyric acid (GABA) receptor beta2 subunit gene (GABRB2) displayed a genotype-dependent decrease in SZ postmortem brain due to abnormal epigentic imprinting of the gene. These findings suggest that DNA modifications might be one of the molecular mechanisms contributing to this abnormal epigenetic phenomenon. As important epigenetics factors, DNA modifications involve 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) that are important markers in the dynamics of DNA methylation and demethylation. To explore potential functional consequences of the epigenetics mechanisms in SZ, the current proposal will examine the relationship of DNA modifications (5hmC and 5mC) and variations (single nucleotide polymorphisms, SNPs) located in the promoter and SZ-associated region of GABRB2 with SZ performed with DNA from postmortem brains of US Caucasian and peripheral white blood cells of Chinese Han. The effects of the above disease-associated DNA modifications and variations on RNA expression were further examined in the postmortem brain samples. Finally, regulations of these functional sites in DNA modification and variation on GABRB2 expression were evaluated in an epigenetic methionine (MET)-induced mouse model of SZ and tissue-cultured cell lines expressing minigene containing disease-associated region or reporter gene containing promoter region. The success of the present project will reveal the synergetic roles of DNA hydroxylmethylation and methylation, DNA variation and RNA expression of GABRB2 involved in the pathophysiology of SZ, and try to identify the genetic and epigenetic biomarkers for monitoring the onset and development of SZ, thus providing novel evidences for early diagnosis of SZ.

遗传和表观遗传等因素诱发的伽玛氨基丁酸能传递下调被认为是精神分裂症（SZ）发病原因之一,但具体机制尚未阐明。我们前期发现SZ尸检脑中伽玛氨基丁酸A型受体beta2亚基基因GABRB2基因型相关的RNA表达下降与基因印迹异常有关，这提示DNA修饰可能介导了这种表观遗传调控异常现象。本项目以DNA修饰过程标志物5甲基胞嘧啶（5mC）和5羟甲基胞嘧啶(5hmC)为切入点，利用临床尸检脑和外周血样本，分析GABRB2疾病易感区和启动子区5hmC/5mC修饰、SNPs变异及其对RNA表达的影响与SZ的相关性；利用蛋氨酸诱导小鼠模型和含易感区的微小基因或启动子的报告基因的细胞模型，检验功能性DNA修饰和变异对基因表达的调控作用。项目完成将揭示GABRB2修饰和变异、RNA表达的协同效应在SZ发生发展中的规律，阐明DNA修饰和基因变异调控RNA表达的分子机制，为寻找监测SZ发生发展生物学标识物提供依据。

遗传和表观遗传等因素诱发的伽玛氨基丁酸能传递下调被认为是精神分裂症（SCZ）发病原因之一,但具体机制尚未阐明。我们前期发现SCZ尸检脑中伽玛氨基丁酸A型受体beta2亚基基因GABRB2基因型相关的RNA表达下降与基因印迹异常有关，这提示DNA修饰可能介导了这种表观遗传调控异常现象。本项目以DNA修饰动态变化过程的标志物5甲基胞嘧啶（5mC）和5羟甲基胞嘧啶(5hmC)为切入点，在临床样本中发现SCZ外周血DNA全基因组5mC和5hmC水平发生异常，并且5hmC与年龄的相关性在疾病与对照组显著不同；通过对GABRB2基因启动子区和AluYi6区的5hmC和5mC修饰水平检测，我们发现了启动子P1区的5hmC/5mC水平与性别、年龄及奥氮平用药史有关系，而P2区的5hmC/5mC水平、AluYi6区5mC水平与家族发病史和齐拉西酮或奥卡西平用药史有关系，且在精神分裂症样本中显著高于对照样本。启动子区5hmC/5mC水平与SNPs rs72815526 (C/A) 和 rs3811997 (C/T)显著相关：在疾病样本中，rs72815526 (C/A) 杂合基因型可显著增加5hmC水平而rs3811997 (C/T)杂合基因型可降低5mC水平；在细胞水平我们发现VPA和AZA处理IMR-32细胞可显著增加GABRB2 mRNA表达水平：AZA增加mRNA表达是通过降低启动子区甲基化，而VPA增加mRNA表达是通过增加Alu区域的组蛋白乙酰化水平。在GABRB2启动子报告基因转染的细胞模型中，改变疾病相关SNP的基因型或CpG位点可显著影响启动子的活性；此外，疾病相关的Alu序列可作为增强子功能来调控GABRB2启动的活性；利用L-methionine诱导构建了斑马鱼社交功能和认知障碍与GABRB2基因关联的行为学模型，并发现斑马鱼中人类GABRB2基因同源的gabrb2基因DNA甲基化修饰和RNA表达存在不同程度的异常，从而模拟和验证了精神分裂症认知和社交功能障碍伴随着GABRB2基因DNA甲基化增加和RNA表达降低。本项研究从临床样本、细胞模型和动物模型三个层次阐述了GABRB2基因的DNA变异、甲基化和羟甲基化修饰对基因表达的调控机制及其在精神分裂症相关行为表型动物模型上的再现，为建立可用于检测疾病发生的在体生物标记提供了依据。